WO2001044442A1 - Arginine-enriched medium composition used for mass-producing recombinant protein in animal cell culture - Google Patents

Arginine-enriched medium composition used for mass-producing recombinant protein in animal cell culture Download PDF

Info

Publication number
WO2001044442A1
WO2001044442A1 PCT/KR2000/001449 KR0001449W WO0144442A1 WO 2001044442 A1 WO2001044442 A1 WO 2001044442A1 KR 0001449 W KR0001449 W KR 0001449W WO 0144442 A1 WO0144442 A1 WO 0144442A1
Authority
WO
WIPO (PCT)
Prior art keywords
medium
cell
medium composition
arginine
concentration
Prior art date
Application number
PCT/KR2000/001449
Other languages
French (fr)
Inventor
Gyun Min Lee
Tae Kyung Kim
Joo Young Chung
Seung Kook Park
Original Assignee
Daewoong Pharm. Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to KR1999/57910 priority Critical
Priority to KR1019990057910A priority patent/KR20010056451A/en
Application filed by Daewoong Pharm. Co., Ltd. filed Critical Daewoong Pharm. Co., Ltd.
Publication of WO2001044442A1 publication Critical patent/WO2001044442A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/524Thrombopoietin, i.e. C-MPL ligand
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018Culture media for cell or tissue culture
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/02Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/30Organic components
    • C12N2500/32Amino acids

Abstract

The present invention relates to an arginine-enriched medium composition used for mass-producing recombinant protein in animal cell culture. Particularly, the present invention relates to the arginine-enriched medium composition containing arginine by the concentration of 160-4200 mg/L which corresponds to 2-50 folds of the previously used medium. The arginine-enriched medium composition of the present invention can be widely used for mass-producing the recombinant protein in animal cell culture by preventing the early growth inhibition of cell and increasing the viability and life span of cell. Especially, the arginine-enriched medium composition is effectively used for mass-producing human thrombopoietin (TPO), human erythropoietin (EPO), blood coagulating agent (tPA) and antibody.

Description

ARGININE-ENRICHED MEDIUM COMPOSITION USED FOR MASS- PRODUCING RECOMBINANT PROTEIN IN ANIMAL CELL CULTURE

FIELD OF THE INVENTION

The present invention relates to an arginine- enriched medium composition used for mass-producing recombinant protein in animal cell culture.

Particularly, the present invention relates to the arginine-enriched medium composition containing arginme by the concentration of 160-4200 mg/L which corresponds to 2-50 folds of the previously used medium.

BACKGROUND

It has been m progress actively study and industrialization which produces monoclonal antibody or therapeutic glycoprotein via mass culture of animal cell as a part of developing medicinal drugs m the field of biotechnology. As a result, it has been developed and marketed about 50 kinds of monoclonal antibodies, EPO and blood coagulating agent. In USA, it has been anticipated that the annual selling amount of biomedicmes derived from animal cells is on an increasing trend of over 15-20& yearly from three billions m 1993 to tourteen billions and five hundred millions m 2003

Especially, m order to maintain the molecular structural stability and the high activity of glycoprotem after administration into the body, its glycosylation and post-transcπptional modification are important. However, since these processes are incomplete or do not perform in microorganisms, it is necessary to use the animal cells for producing the biologically active form of glycoprotem.

The representative glycoprotem recently used for the medical world is thrombopoietm (hereinafter, as referred to be λTPO' ) , erythropoietm (hereinafter, as referred to be ΛEPO' ) and so on. TPO, produced in liver or kidney, regulates the number of the platelet and is used for therapy of thrombocytopenia that occurs by cancer treatment, bone marrow transplantation or any other causes .

Particularly, megakaryocytic progenitor cells existed in the bone marrow cells, are destroyed by cancer treatment or in the procedure of bone marrow transplantation, the number of platelet decrease, and the thrombocytopenia occurs .

Throiϊioocytopenia is early induced bleeding by light external stimulus and without external stimulus m case of serious thrombocytopenia. Once bleeding is induced, it is difficult to stop, so thrombocytopenia

? becomes a serious problem causing deaths by severe bleeding .

As a method for treatment of the thrombocytopenia up to now, blood transfusion of the platelet is solely used. However, it has also problems that the donors of blood needed for platelet transfusion are lack, and the infection such as AIDS virus or hepatitis virus derived from blood and immune response according to foreign platelet transfusion occurs.

Platelet is a blood composition produced from megakaryocytic progenitor cells and has a function that inhibits bleeding. The number of platelet is regulated by TPO glycoprotem. TPO stimulates the proliferation and reproduction of megakaryocytic progenitor cells in bone marrow cell leading to the production of circulating platelets (Lok et al . , Na t ure, 369:565-568, 1994; De Savage et al . , Na t ure, 369:533-568, 1994).

After human thrombopoietm (hTPO) cDNA among TPO genes is cloned for the first time at 1994 (Lok et al . , Na t ure , 369:565-568, 1994; De Savage et al., Na turer 369:533-568, 1994; iyazaki et al . , Exp . Hema tol . , 22:838, 1994; WO 95/18858), hTPO is under clinical trials for treatment of thrombocytopenia by the property cf hTPO whιch regulates the numoer of platelet (Murray et d . , E . ->ema tol . , 26:207-216, 1998). From this, it has been the increasing need for mass- production of TPO.

CHO cell among the animal cells is the most widely used host since it has advantages of the stable gene transfection, high productivity and rapid adaptability to serum free medium. Especially, CHO-DUKX cells with deletion of dhfr gene is able to use dhfr gene as a selection marker and increase the gene expression by gene amplification process via methothrexate (MTX) , and used for expression and production of various therapeutic proteins.

Cells are easily affected by external mechanical force since they only consist of plasma membrane m the absence of cell wall, and their growth and metabolic activity are seriously inhibited by a large amount of lactic acid generated by conversion of carbon and energy source during metabolism, and ammonia made form glutamme, which is supplied as energy source. Generally, since the cells which produce foreign protein by gene transfection needs more gene replication, transcription, and protein synthesis compared with the cell which is not so, its growth becomes slower and slower (Beutley et al . , Bi otecn . Bioeng . , 35:668, 1990) .

Especially, it has been reported that the more the productivity increases by amplification of gene which expresses foreign protein by MTX, the more the growth rate decreases severely (Gu et al . , Cytotech . , 18:159, 1996) . In serious case, cell death occurs early m the cultivation by inhibiting the cell growth (Sigh et al . , Biotech , Bioeng. , 44:720, 1994).

As a result, the protein concentration produced by the cell is m the low level, and actually, we demonstrated that cell growth inhibition occurs during the batch culture of CHO cell lines that produce TPO and EPO antibody, respectively.

Since the cell growth inhibition is the major cause of low productivity, it is necessary to establish the optimal culture condition to prevent the growth inhibition, and it is the most important for optimizing the medium composition by increasing osmotic pressure of medium or adding sodium butyrate into the medium. These methods induce the increase of the expression rate of target protein, but they still have the disadvantage to reduce cell viability drastically.

As the nutrient reguirements for animal cell are very complex, it is essential for adding ammo acid and vitamin into the medium, and it also require serum containing many growth factors . These study about the medium composition have been performed mainly relation to the preparation of serum free medium. arious medium additives are developed to increase the cell viability during serum free culture, and metabolic engineering approach is also under active research. Recently, the study of ammo acid role in relation to the programmed cell death hybridoma has been actively going on (Franek and Sramkova, Cytotech . , 23:231, 1997; Mercille and assie, Biotech . Bioeng. , 44:1140, 1994), and it has been made efforts to prevent the cell death by adding the various ammo acid complex or ammo acid analogues into the medium. Particularly, it has been reported in case of treating glutamme to CHO cell (Sanfeliu et al . Biotecnnol . Bioeng. , 64:46, 1996); glycme, alanme serme, threonme, proline, asparagme, glutamme histidme ( Franek and Sramkova, Cytotech . , 23:23, 1997) glutamme ( Franek, Biotech . Bi oeng . , 45:86, 1995) methion e (Perreault et al . , Cytotech . , 13:99, 1994) to hybridoma; and cysteme (Mercille et al . , Biotech . Bioeng. , 44:1140, 1994) to cancer cell.

However, there is no report that argm e-enriched medium induces the high-level production of the recombinant protein.

In the production process of TPO, the above- mentioned early cell growth inhibition is serious problem to the increase of productivity of the protein. In addition, it brings a severe problem to the development or cell line. Especially, since the cell death frequently occurs during MTX treatment by the high concentration for gene amplification, it is difficult to maintain cell culture for cell line development .

To overcome these problems, the present inventors have studied the medium composition that can induce the high-level production of the recombinant protein with m animal cell culture.

As a result, the present inventors have found that life span and viability of cells are increased by adding 160-4200 mg/L of arginine to the medium, and the high-level production of recombinant protein can be accomplished therefrom.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a medium composition which is used for mass-producing the recombinant protein via animal cell culture by preventing the early cell growth inhibition and increasing the cell viability.

In accordance with the present invention, the rorego g objects and advantages are readily obtained. The present invention provides the argin e- enriched medium used for animal cell culture.

The 'arginine-enriched medium' in the present invention means the medium adding the high concentration of arginme to the basal medium.

The 'basal medium' means the well-known medium used for animal cell culture, and can be selected from the group of DMEM (Dulbecco's modified eagle's medium), MEM (minimum essential medium eagle), RPMI 1640 medium, medium 199, DMEM/F12, IMDM (Iscove's modified Dulbecco' s medium) , but is not restricted within the group. The well-known media for animal cell culture or their modified mediums are used for mass-producing the recombinant protein in the present invention.

Further features of the present invention will appear hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cell growth state in the arginine- enriched medium composition of the present invention according to the argmine concentration, wherein

-• - ; basal IMDM (Iscove's modified Dulbecco's medium1 containing 84 mg/L of argmine; -I - ; IMDM containing 168 mg/L of argmine;

-▼ - ; IMDM containing 420 mg/L of argmine. FIG. 2 shows a production rate of TPO the arginine-enriched medium composition of the present invention according to the concentration of argmme, wherein -• -; basal IMDM (Iscove's modified Dulbecco's medium) containing 84 mg/L of argmme;

-I -; IMDM containing 168 mg/L of argm e;

-▼ -; IMDM containing 420 mg/L of argmme.

FIG. 3 shows a viability of cell in the arginine- enriched medium composition of the present invention according to the concentration of argmme.

FIG. 4 shows a viability of cell represented by measuring the relative MTT value m the arginine- enriched medium composition that contains the other ammo acids except argmme in the concentration of 3 folds to the basal medium.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail .

In one aspect, the present invention provides the medium composition containing the high concentration of argmine for increasing the production rate of TPC recomoinant protein b\ preventing the early cell growth inhibition and increasing the maximum number of viable cell on the animal cell culture for mass-producing the recombinant protein.

The medium composition of the present invention is prepared by adding a concentrated argmme solution in concentration of 160-4200 mg/L to the basal medium in concentration of 70-200 mg/L.

In a preferred embodiment, the present inventors examine the effects of various ammo acids and other nutrients for the production of recombinant protein m animal cell culture by adding them to the basal culture medium. As a result, it is found that argmme has an excellent effect for preventing the early cell growth inhibition beyond compared with the other ingredients. This result may be caused that the high concentration of argm e prevents the programmed cell death appeared in the production of recombinant protein by animal cell culture, or the added amount of argmme is enough for compensating the argmme consumption which is needed for the production of recombinant protein. In addition, since argmme has a high stability and solubility to the medium, shows no toxicity to cells and is relatively low in price, the argmme-enriched medium composition is very effective for cell culture process. The recombinant protein produced by using the medium composition of the present invention includes colon /-stimulating factor, blood factor, growth hormone, interferon, enzyme protein, antibody and so on.

It is preferable to use the medium composition for the production of glycoprotein like TPO, EPO or blood coagulating agent, especially used for increasing the production rate of TPO.

The medium composition of the present invention is also used for animal cell culture which is widely used for the production of recombinant protein, and is preferable to use for culture of CHO, kidney cell derived from human, monkey, hamster such as BHK-21 (baby hamster kidney), Hela cell, NIH 3T3 cell, rat embryo fibroblast cell, and more preferable to use for culture of CHO dhfr mutant.

In another preferable embodiment of the present invention, the arginine-enriched medium composition is used for the production of TPO by culturing CHO DUKX Bll cell. The medium composition of the present invention can be prepared by adding the concentrated arginme solution to the general medium such as DMEM or MEM used for animal cell culture or the modified medium such as IMDM by the concentration of 160-4200 mg/L.

It is possible to regulate the proper concentration of argmme m the medium by adding the concentrated arg me solution. The more the medium contains argmme by the concentration between 2 folds and 5C folds, the more the number of viable cell increases m proportion to the argmme concentration.

However, m case of exceeding 50 folds, it reaches the saturated concentration, and the number of viable cell does not increase any more. But, although the argmme concentration exceeds 50 folds, there is no toxicity to cells, and the osmotic pressure by argmme lonization is insignificant. In addition, as a result of comparing the absence of argmme with the presence of argmme, there is no difference in the consumption/release pattern of total ammo acid, glucose and lactate.

Namely, no remarkable physiological changes occurred in cells. Consequently, it is preferable to add argmme by the concentration of 2 to 50 folds in the side of critical concentration related to the argmme effect. When the relationship between the added amount of argmme and the argmme effect is more 10 to 20 folds of argmme than the basal medium, the concentrated argmme solution is prepared by dissolving 16.8-84 mg/L of high concentration of argmme in distilled water, and can be stored below - 20 °C.

In order to prevent the early cell growth inhibition, it is preferable to change the cultured medium mtc the medium composition of the present invention during cultivation or add the proper amount of concentrated argmme solution to the cultured medium before the cell die. It is more preferable to culture by using the medium composition of the present invention from the beginning of the cultivation.

The present invention has examined the cell growth condition according to the argmme concentration n the medium. It is confirmed that in case of culturing in IMDM containing argm e in the concentration of 84- 420 mg/L, the cell growth rate increases in proportion to the argmme concentration. As a result of examining the produced amount of TPO according to the argmme concentration in the medium, it is found that the amount of TPO produced by culturing in the medium containing 84-420 mg/L of argmme, increases maximum up to 4.3 folds compared with the basal medium m proportion to the argmme concentration m the medium (see FIG. 2) .

When argmme is added by the concentration of 2- 100 folds compared with the basal medium in order to measure the viable cell number according to the argmme concentration in the medium, but it does not increase any more above 50 folds.

This result represents that argmme shows the effect maintaining m proportion to the atgmme concentration up to 50 folds compared with the basal medium, but arg me does not show any effect required for the cell ziabilitv since its concentration is to be saturated abo/e 5U folds. EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples . However, it will be appreciated that those skilled m the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1 : Growth of animal cell in arginine-enriched

medium composition

Transformant WF21 producing TPO was prepared by transforming CHO dhfr mutant cell (ATCC CRL 9096) with pDCT (KRP 99-25143) , and was used for animal cell line for producing TPO recombinant protein.

WF21 cell was inoculated into 3 ml of medium on 6 well plate (Nunc, Denmark) by the concentration of 2 X 105 cells/well. The medium used a basal IMDM (Gibco BRL, USA) containing 84 mg/L of argm e (Sigma, USA), IMDM containing 168 mg/L of argmme by 2 folds compared with the nasal medium, and IMDM containing 420 mg/L of argmme by 5 folds compared with the basal medium, and 10 dFBS (dialyzed fetal bovine serum) and 20 nM MTX was added into the each medium.

The inoculated cells were cultured m the incubator (Forma Scientific, USA) used for the animal cell culture in the condition of 37 °C, 5% C02 for 5 days by batch culture. The number of cell was measured by Trypan blue dye exclusion method (Freshney, Culture of animal cells, 268-270, 1994) using hemocytometer (Hausser Scientific, USA) .

As a result, when the cells were cultured in the basal IMDM containing 84 mg/L of argmme, the number of cells reached to the maximum concentration of 3.1 X lO5 cells/well on 2 days after beginning of the cultivation and immediately decreased. On the other hand, when the cells were cultured in the basal IMDM containing 168 mg/L of argm e, it increased to the maximum concentration of 5.9 X 105 cells/well on 3 days after beginning of the cultivation and decreased. In case of IMDM containing 420 mg/L of argmme, the number of cells was still remained at its high concentration to 1.5 X 106 cells/well without decreasing the number of cell on 5 days (FIG. 1) .

Comparative Example 1 : Growth of animal cell in the basal medium composition

Except that the basal medium containing 84 mg/L of argmme was used, WF21 cell line was cultured by the same method described in the Example 1 (FIG. 1) .

From the result of Comparative Example 1, it was demonstrated that the use of argmme-enriched medium for animal cell culture made the life span of cell longer and the maximum concentration of cells higher.

Example 2 : Production of recombinant protein in the arginine-enriched medium composition

WF21 cell line was performed by batch culture using the basal IMDM, IMDM containing 168 mg/L of arg me and IMDM containing 420 mg/L of argm e respectively, by the same method described m the Example 1. After that, the concentration of TPO produced by WF21 cell line was analyzed by ELISA (sandwich enzyme- linked lmmunosorbent assay) method. A mouse monoclonal antibody to human TPO (hTPO) (R&D System, USA) was used as a coating antigen, and anti-hTPO monoclonal antibody labeled with biotin was used as a secondary antibody. A coloring reaction was performed by using Streptavidme-peroxidase (Sigma, USA) as a coloring enzyme and TMB (3, 3' , 5, 5 ' -Tetramethyl-benzidme base, GibcoBRL, USA) as a substrate of this enzyme, and stopped by adding 1 M sulfuric acid solution. A selling TPO was used as a standard by quantifying its concentration using TPO ELISA kit (Quantιkmeτ , R&D system, USA) and diluting its concentration into 10, 5, 2.5, 1.25 and 0.625 ng/ml. The degree of coloring was measured at 450 nm by using a microplate reader (Model 550, BioRad, USA), and described m the Table 1.

<Table 1> the variation of TPO concentration according to the argmme concentration

Figure imgf000019_0001

As described in the Table 1, the concentration of TPO produced by culturing in the medium containing 168 mg/L of argmme was about 2.1 folds higher than that by culturing in the basal medium, and the concentration of TPO produced by culturing the medium containing 420 mg/L of argmme about 4.3 folds higher than by culturing in the basal medium (FIG. 2) .

Comparative Example 2 : Production of recombinant protein in the basal medium

Except that the basal IMDM containing 84 mg/L of argm e was used, the cells were cultured m the same condition described ir the Example 2 and ELISA analysis was performed oy the same method described in the Example 2.

As shown m the Table 1, it was confirmed that when the recombinant protein was produced by using the animal cell, the use of argmme-enriched medium composition in the concentration range of 168 to 420 mg/L as the starting medium made the production rate increased up to 4.3 folds compared with the use of basal medium containing 84 mg/L of argmme.

Example 3 : Relationship between the arginine concentration and the number of viable cell

After filling 3 ml of IMDM basal medium in 6-well plate, the concentrated argmine solution was added to the well to be containing argmme by the concentration of 1, 2, 5, 10, 20, 50 and 100 folds compared with the basal medium, respectively. The cells were inoculated into the each well by the concentration of 2 X 105 cells/well and cultured for 9 days. After 9 days, the number of viable cell was measured by the same method described in the Example 1 (FIG. 3), and described in the Table 2.

<Table 2> The number of viable cell according to the argmine concentration

Figure imgf000021_0001

As a result, it was demonstrated that when the arg me concentration was to be up to 50 folds compared with the basal medium, the number of viable cell increased m the proportion to the argmme concentration, but the number of viable cell did not increase m above 50 folds of argmme concentration.

Comparative Example 3 : Variation of maximum cell concentration according to the enriched materials in the medium

WF21 cell line was cultured by using alanine, asparagme, glutamme, glycine, histidme, proline, seπne, isoleucme, lycme, methionme, phenylalanme, threonme, tryptophane, tyrosme and valme as the enriched materials in the medium via the same method described m the Example 1.

The maximum number of viable cell using the enriched medium with the above materials was compared with that using the argmme-enriched medium by measuring the relative value of MTT (3- [4, 5- Dιmethylthιazol-2-yl] -2, 5-dιphenyltetrazolιum bromide, Thiazolyly blue) (FIG. 4) .

Particularly, the number of viable cell was analyzed by adding the above ammo acids into IMDM by 3 X concentration of the basal medium; adding 5 mg/ml of MTT into IMDM after 5 days; culturing at the same concentration for 4 hours; and measuring absorbance at 540 nm as the relative MTT value by using the Microplate Reader (Model 550, BioRad, USA) , and described in the Table 3.

<Table 3> Variation of the maximum cell concentration according to the enriched materials m the medium

Figure imgf000022_0001
Figure imgf000023_0001

As described in the Table 3, when the MTT value in case of the arginine-enriched medium was compared with that in case of the enriched-medium with any other amino acids, it was found that arginine represented significantly high MTT value. Therefore, in case of the arginine-enriched medium, the number of viable cell increased by far, and the recombinant protein was obtained in a high concentration.

INDUSTRIAL APPLICABILITY

The present invention provides an arginine- enriched medium composition used for mass-producing the recombinant protein via animal cell culture. The arginine-enriched medium composition of the present invention is effectively used for the production of recombinant protein on a high concentration compared with the existing basal medium by preventing the early growth inhibition of animal cell and increasing the maximum concentration of cell via promotion of cell viability .

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

??

Claims

What is Claimed is
1. A medium composition for animal cell culture containing 160-4200 mg/L of argmme m the basal medium that is used for mass-production of recombinant protein.
2. The medium composition for animal cell culture according to the claim 1, wherein the recombinant protein is selected from the group of thrombopoietin (TPO), erythropoietm (EPOj, blood coagulating agent (tPA) and antibodies.
3. The medium composition for animal cell culture according to the claim 1, wherein the basal medium is selected from the group of DMEM, MEM, RPMI 1640 medium, medium 199, DMEM/F12 and IMDM.
4. The medium composition for animal cell culture according to the claim 1, wherein the animal cell is selected from the group of CHO cell, BHK cell, Hela cell, NIH 3T3 cell and rat embryo cell.
5. The medium composition for animal cell culture according to the claim 4, wherein the CHO cell is
CHO dhrr mutant. The medium composition for animal cell culture according to the claim 1, wherein the medium composition is comprised of glucose 1-4.5 g/L, arginine 0.07-0.2 g/L, glutamine 0.1-0.6 g/L and the other amino acids 0.001-0.15 g/L.
PCT/KR2000/001449 1999-12-15 2000-12-13 Arginine-enriched medium composition used for mass-producing recombinant protein in animal cell culture WO2001044442A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1999/57910 1999-12-15
KR1019990057910A KR20010056451A (en) 1999-12-15 1999-12-15 Arginine-enriched medium used for mass-producing recombinant protein in animal cell culture

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
AU20286/01A AU2028601A (en) 1999-12-15 2000-12-13 Arginine-enriched medium composition used for mass-producing recombinant proteinin animal cell culture

Publications (1)

Publication Number Publication Date
WO2001044442A1 true WO2001044442A1 (en) 2001-06-21

Family

ID=19626045

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2000/001449 WO2001044442A1 (en) 1999-12-15 2000-12-13 Arginine-enriched medium composition used for mass-producing recombinant protein in animal cell culture

Country Status (3)

Country Link
KR (1) KR20010056451A (en)
AU (1) AU2028601A (en)
WO (1) WO2001044442A1 (en)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013009648A2 (en) 2011-07-08 2013-01-17 Momenta Pharmaceuticals, Inc. Cell culture process
CN103484426A (en) * 2013-10-15 2014-01-01 齐鲁制药有限公司 Non-animal source low-protein culture medium
US8921526B2 (en) 2013-03-14 2014-12-30 Abbvie, Inc. Mutated anti-TNFα antibodies and methods of their use
US8946395B1 (en) 2013-10-18 2015-02-03 Abbvie Inc. Purification of proteins using hydrophobic interaction chromatography
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9062106B2 (en) 2011-04-27 2015-06-23 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9150645B2 (en) 2012-04-20 2015-10-06 Abbvie, Inc. Cell culture methods to reduce acidic species
US9181572B2 (en) 2012-04-20 2015-11-10 Abbvie, Inc. Methods to modulate lysine variant distribution
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9193787B2 (en) 2012-04-20 2015-11-24 Abbvie Inc. Human antibodies that bind human TNF-alpha and methods of preparing the same
US9206390B2 (en) 2012-09-02 2015-12-08 Abbvie, Inc. Methods to control protein heterogeneity
US9234033B2 (en) 2012-09-02 2016-01-12 Abbvie, Inc. Methods to control protein heterogeneity
US9249182B2 (en) 2012-05-24 2016-02-02 Abbvie, Inc. Purification of antibodies using hydrophobic interaction chromatography
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
US9550826B2 (en) 2013-11-15 2017-01-24 Abbvie Inc. Glycoengineered binding protein compositions
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6251983A (en) * 1985-09-02 1987-03-06 Hagiwara Yoshihide Serum-free culture medium for cultivating human/human hybridoma
DD265425A1 (en) * 1985-09-04 1989-03-01 Friedrich Loeffler Inst Insel culture media methods for manufacturing ph stable
EP0481791A2 (en) * 1990-10-17 1992-04-22 The Wellcome Foundation Limited Culture medium for CHO-cells and adapted CHO-cells
EP0725140A1 (en) * 1995-01-31 1996-08-07 Sanofi Process of extraction of periplasmic proteins from prokaryotic microorganisms in the presence of arginine
WO1998004681A2 (en) * 1996-07-25 1998-02-05 Genzyme Corporation Chondrocyte media formulations and culture procedures

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6251983A (en) * 1985-09-02 1987-03-06 Hagiwara Yoshihide Serum-free culture medium for cultivating human/human hybridoma
DD265425A1 (en) * 1985-09-04 1989-03-01 Friedrich Loeffler Inst Insel culture media methods for manufacturing ph stable
EP0481791A2 (en) * 1990-10-17 1992-04-22 The Wellcome Foundation Limited Culture medium for CHO-cells and adapted CHO-cells
EP0725140A1 (en) * 1995-01-31 1996-08-07 Sanofi Process of extraction of periplasmic proteins from prokaryotic microorganisms in the presence of arginine
WO1998004681A2 (en) * 1996-07-25 1998-02-05 Genzyme Corporation Chondrocyte media formulations and culture procedures

Cited By (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9090688B2 (en) 2011-04-27 2015-07-28 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9505834B2 (en) 2011-04-27 2016-11-29 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9062106B2 (en) 2011-04-27 2015-06-23 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9365645B1 (en) 2011-04-27 2016-06-14 Abbvie, Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9255143B2 (en) 2011-04-27 2016-02-09 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US8852889B2 (en) * 2011-07-08 2014-10-07 Momenta Pharmaceuticals, Inc. Cell culture process
WO2013009648A2 (en) 2011-07-08 2013-01-17 Momenta Pharmaceuticals, Inc. Cell culture process
US9914951B2 (en) 2011-07-08 2018-03-13 Momenta Pharmaceuticals, Inc. Cell culture process
CN103717729A (en) * 2011-07-08 2014-04-09 动量制药公司 Cell culture process
EP2729561A4 (en) * 2011-07-08 2015-06-24 Momenta Pharmaceuticals Inc Cell culture process
CN103717729B (en) * 2011-07-08 2017-11-21 动量制药公司 The cell culture method
US9475858B2 (en) 2011-07-08 2016-10-25 Momenta Pharmaceuticals, Inc. Cell culture process
US9957318B2 (en) 2012-04-20 2018-05-01 Abbvie Inc. Protein purification methods to reduce acidic species
US9150645B2 (en) 2012-04-20 2015-10-06 Abbvie, Inc. Cell culture methods to reduce acidic species
US9181572B2 (en) 2012-04-20 2015-11-10 Abbvie, Inc. Methods to modulate lysine variant distribution
US9346879B2 (en) 2012-04-20 2016-05-24 Abbvie Inc. Protein purification methods to reduce acidic species
US9193787B2 (en) 2012-04-20 2015-11-24 Abbvie Inc. Human antibodies that bind human TNF-alpha and methods of preparing the same
US9708400B2 (en) 2012-04-20 2017-07-18 Abbvie, Inc. Methods to modulate lysine variant distribution
US9359434B2 (en) 2012-04-20 2016-06-07 Abbvie, Inc. Cell culture methods to reduce acidic species
US9334319B2 (en) 2012-04-20 2016-05-10 Abbvie Inc. Low acidic species compositions
US9505833B2 (en) 2012-04-20 2016-11-29 Abbvie Inc. Human antibodies that bind human TNF-alpha and methods of preparing the same
US9683033B2 (en) 2012-04-20 2017-06-20 Abbvie, Inc. Cell culture methods to reduce acidic species
US9249182B2 (en) 2012-05-24 2016-02-02 Abbvie, Inc. Purification of antibodies using hydrophobic interaction chromatography
US9290568B2 (en) 2012-09-02 2016-03-22 Abbvie, Inc. Methods to control protein heterogeneity
US9234033B2 (en) 2012-09-02 2016-01-12 Abbvie, Inc. Methods to control protein heterogeneity
US9206390B2 (en) 2012-09-02 2015-12-08 Abbvie, Inc. Methods to control protein heterogeneity
US9512214B2 (en) 2012-09-02 2016-12-06 Abbvie, Inc. Methods to control protein heterogeneity
US9708399B2 (en) 2013-03-14 2017-07-18 Abbvie, Inc. Protein purification using displacement chromatography
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
US8921526B2 (en) 2013-03-14 2014-12-30 Abbvie, Inc. Mutated anti-TNFα antibodies and methods of their use
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins
CN103484426A (en) * 2013-10-15 2014-01-01 齐鲁制药有限公司 Non-animal source low-protein culture medium
CN103484426B (en) * 2013-10-15 2014-11-26 齐鲁制药有限公司 Non-animal source low-protein culture medium
US9266949B2 (en) 2013-10-18 2016-02-23 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9688752B2 (en) 2013-10-18 2017-06-27 Abbvie Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9522953B2 (en) 2013-10-18 2016-12-20 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9200069B2 (en) 2013-10-18 2015-12-01 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9315574B2 (en) 2013-10-18 2016-04-19 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US8946395B1 (en) 2013-10-18 2015-02-03 Abbvie Inc. Purification of proteins using hydrophobic interaction chromatography
US9499616B2 (en) 2013-10-18 2016-11-22 Abbvie Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9200070B2 (en) 2013-10-18 2015-12-01 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9550826B2 (en) 2013-11-15 2017-01-24 Abbvie Inc. Glycoengineered binding protein compositions

Also Published As

Publication number Publication date
KR20010056451A (en) 2001-07-04
AU2028601A (en) 2001-06-25

Similar Documents

Publication Publication Date Title
Fogolın et al. Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells
US8663945B2 (en) Methods of producing anti-TNF-alpha antibodies in mammalian cell culture
DE69635076T2 (en) A method of monitoring, manipulating the sialylation of produced by mammalian cell culture protein
CA2053586C (en) Defined culture medium for cho cells
ES2252876T5 (en) Process for preparing polypeptides with proper glycosylation
EP0066284A2 (en) Culture medium and its use
US20090111143A1 (en) Methods and Compositions for Mammalian Cell Lines for Transfection and Protein Expression in Serum-Free Medium
CA2536239C (en) Method for recloning production cells
ES2285188T3 (en) Method and means for controlling sialylation of proteins produced by mammalian cells.
US6406909B1 (en) Serum-free medium for culturing animal cells
CA2578141C (en) Production of polypeptides
CN101297027B (en) Methods of protein production using anti-senescence compounds
ES2625045T3 (en) Culturing mammalian cells
CA2578137C (en) Production of .alpha.-abeta
US20100221823A1 (en) Method for culturing mammalian cells to improve recombinant protein production
AU2008242633B2 (en) Methods of protein production using anti-senescence compounds
JP5401319B2 (en) Glycolysis inhibitors in cell cultures
US5856179A (en) Polypeptide production in animal cell culture
CN102858952B (en) Improved cell culture medium
EP2563906B1 (en) Process for cultivation of cho cells
US9388447B2 (en) Method for culturing mammalian cells to improve recombinant protein production
CA2578138C (en) Production of tnfr-ig fusion protein
EP2601287B1 (en) Dipeptides to enhance yield and viability from cell cultures
ES2557741T3 (en) Methods for increasing production of polypeptides
US6924124B1 (en) Feeding strategies for cell culture

Legal Events

Date Code Title Description
AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP